The invention relates to luminescence optical microscopy and particularly to an apparatus and method for selectively optically exciting luminescence in particular zones in a specimen such as a biological cell or tissue.
Optical microscopy using fluorescence tagging for the determination of three-dimensional structure of cells and tissues is an important diagnostic and research procedure. There are a great number of dyes which can be attached to various structures within the cell. When excited by a particular wavelength of light these dyes will fluoresce or phosphoresce. For example, several common dyes will emit a red glow when excited by green light. Hence, one can see structures to which a fluorescent dye is attached. The presence and location of the tagged structures can provide important diagnostic and structural information for basic research and clinical diagnostics.
Fluorescence imaging, in particular is of vast utility in cell biology because of the high specificity of fluorescently labeled protein analogs, antibodies, hybridization probes, enzyme substrates, lipid analogs, and peptides, as well as stains. Fluorescence micrographs of extremely complicated objects such as intact cells typically show clearly the distribution of the tracer molecules, all other components being "invisible". The important optical characteristics of many biological specimens that allow for this simple interpretation of the image field is that cells are generally weakly refractive and weakly absorptive objects.
Conventional microscope images contain information about the 3-D structure of the object when the depth of field of the lens system is smaller than the axial dimension of the specimen. This means that in a single image, the axial location of a particular feature is encoded by its degree of defocus. A 3-D image data set, which is a "zero-order" estimate of the true structure of the object, is obtained by recording a series of images as the object is stepped through the focal plane of the microscope, a procedure known as optical sectioning microscopy (OSM). Each image is a spatially filtered axial projection of the object, and each generally contains in-focus and out-of-focus features. One of the central problems in 3-D microscopy is the removal of out-of-focus features from the 3-D image by optical and image processing methods thereby deriving a refined estimate of the true object.
In fluorescence microscopy there is a linear relationship between the emitter distribution in the object and the intensity distribution in the image field. This is caused by the mutual incoherence of fluorescence emission. Dye molecules in the specimen radiate independently so that the individual intensity fields are simply superimposed in the image plane. It is possible to deconvolve the 3-D image to attenuate the out-of-focus portions. However, for the various methods that have been proposed there is a trade-off of recovery of high-resolution structure for accuracy or stability.
The alternative to computational refinement of optical sectioning image data is confocal scanning fluorescence microscopy (CSFM) in which direct optical spatial filtering is used to remove out-of-focus light waves from the detector field. In one version of this type of instrument fluorescence is excited in the specimen by a highly focused beam. In the image plane of the microscope a pin hole is placed at the point optically conjugate to the focal point of the beam and a high gain, low noise detector is placed behind the pin hole. The microscope acts as a spatial filter that detects efficiently only fluorescence photons that originate near the beam focus. 3-D image data is obtained by raster scanning of the beam relative to the specimen, either optically or mechanically, and stepping the specimen axially through the focal plane to get stacked images. Confocal methods have several shortcomings. For example, such images often have a low signal to noise ratio. Hence, the resolution of the image is often severely compromised. Also, scanning usually is relatively slow, with scan times up to 64 seconds per frame for high signal-to-noise images. Indeed, there are many circumstances in which this technique cannot be utilized.
For fundamental reasons, a fluorescence microscope is more severely limited in axial (depth, or inter-image plane) resolution, as opposed to transverse (in the image plane) resolution. Consider a microscope with a lens having a high numerical aperture (NA) and a specimen of refractive index n illuminated by a light beam having a wavelength .lambda.. The well-known Rayleigh resolution formula, 0.61.lambda./(NA), sets transverse resolution at about 0.2 .mu.m via direct imaging. This can be halved, in principle, by confocal scanning. In comparison, the axial equivalent of the Rayleigh formula, 2n.lambda./(NA).sup.2, is in the range 0.7-0.9 .mu.m, typical for high-quality fluorescence OSM image sets. Computational image processing or confocal scanning can reduce this to 0.4-0.5 .mu.m. A more restrictive analysis, the Rayleigh quarter-wave criterion, .lambda./8n sin.sup.2 (1/2 sin.sup.1 NA/n), gives a theoretical axial resolution in the range 0.13-0.17 .mu.m for the best microscope lenses. This has been demonstrated in transmitted light microscopy, but not in fluorescence, due to the lack of mutual coherence in fluorescence imaging, and the generally lower signal-to-noise level. Therefore when the specimen contains fine stratified structural features, or simply when it is thinner than the depth of field, fluorescence OSM or even CSFM is unable to yield significant 3D information.
U.S. Pat. No. 4,621,911 discloses a method and apparatus called standing wave luminescence or fluorescence microscopy (SWFM) in which a specimen is illuminated in a fluorescence microscope by means of a standing wave field at the excitation wavelength. This field is preferably produced by crossing two equal amplitude coherent beams from a laser. The direction of the beams is such that the nodal and anti-nodal planes in this field are parallel to the object plane of the microscope. Under this condition fluorescence is excited in laminar zones in the specimen, maximally at the location of each anti-nodal plane. One of these planes can be made coincident with the in-focus plane. In this way in-focus features of the specimen are made brightly fluorescent. Immediately adjacent features above and below the in focus plane are in nodal zones and are, therefore, only weakly fluorescent.
U.S. Pat. No. 4,621,911 teaches a theory and embodiments for creation and manipulation of a periodic standing wave field superimposed with the specimen in a fluorescence microscope, and that sets of images obtained by standing wave excitation contain Fourier coefficient information on the axial (depth) structure of the object down to an axial resolution limit of .lambda./4n, as small as 0.068 .mu.m. The embodiments of the patent include several methods for producing a standing wave field by crossing two equal-amplitude collimated s-polarized coherent beams at complementary angles relative to the axis of the microscope. These embodiments include the use of total internal reflection (TIR), a mirror or prism, or a wavelength-selective high reflector to fold a laser beam in the specimen region of the microscope, independent coherent beams entering the specimen from opposite sides, or a re-entrant beam that first emerges from the objective lens into the specimen. The patent also includes embodiments where the nodal planes are not parallel to the object focal plane although the parallel condition is of principal interest here. Nevertheless, the method and apparatus of the '911 patent do not overcome the problems associated with the presence of out of focus luminescent portions in the image.